Study On The Toxicity Of Yeast Active Peptide Intervention Of DON To IPEC-J2

Under normal circumstances,the cell maintains a redox balance.When a large amount of free radicals accumulate in the body,oxidative stress occurs.Studies have shown that deoxynivalenol(DON)induction is induced by reactive oxygen species(ROS).Oxidative stress is considered to be one of its mechanisms leading to cell death.Yeast cell wall extracts have been reported to have good antioxidant activity.Based on the previous research on yeast cell wall extracts interfering with DON cytotoxicity in this laboratory,this experiment extracted components with strong antioxidant activity from Brady yeast And,from the perspective of anti-oxidation in pig intestinal epithelial cells,explore the intervention effect of yeast enzymatic hydrolysate on DON-induced cellular oxidative stress at the molecular level.1.Extraction of yeast enzymatic hydrolysis active peptides and identification of their active ingredients:Using Blastocystis as raw materials,a class of substances with strong antioxidant properties are extracted from yeast by enzymatic hydrolysis.The single factor experiment and orthogonal experiment were used to explore the best scheme for extracting the enzymatic hydrolysate of yeast,and through a series of experiments to isolate,purify,analyze and identify the enzymatic antioxidant of the yeast,the results showed:(1)Using the extraction rate of peptides and the clearance rate of DPPH free radicals as research indicators,a single factor orthogonal test was used to optimize the extraction method of yeast actives,and the optimal scheme was obtained:temperature40?,p H value 7,The reaction time was 15h.At this time,the peptide extraction rate was 15.26%,and the DPPH radical scavenging rate was 39.54%.(2)Gel permeation chromatography analysis of the average molecular weight of yeast enzymolysis actives was 756.5 Da.Ultraviolet spectroscopy,infrared spectroscopy and amino acid analyzer detected that the substance was a class of antioxidant short peptides.(3)Sephadex G-15 chromatography column was used to separate YE-4,which has the strongest antioxidant activity of DPPH free radicals IC₅₀=0.05?g/m L.(4)Analyze the peptide sequence of YE-4 by LC-MS/MS to obtain the six largest possible peptides:Val-Glu-Leu-Lys-Leu-Gln(728.46Da),Lys-Pro-Glu-Leu-Arg(683.4 Da),Met-Val-Val-Leu-Arg(616.38 Da),Phe-Asp-Asp-Tyr-Arg-Pro-Gln(937.45 Da),Tyr-Leu-Gly-Tyr-Leu-Gln-Leu-Leu-Arg(1266.7 Da),Leu-Lys-Glu-Gln-Phe-Glu(792.41 Da).2.Evaluation of the antioxidant properties of the yeast active peptides:the antioxidant activity of the yeast active peptides was evaluated from multiple angles such as free radical scavenging,reducing ability determination,metal ion chelation ability determination and its application in the H₂O₂-Saccharomyces cerevisiae oxidation model.Property evaluation,the results show:(1)Yeast active peptides have a good scavenging effect on hydroxyl radicals,DPPH radicals,ABTS radicals and superoxide anion radicals.The half-clearing rate/half-suppression rate IC₅₀of several free radicals are:hydroxyl Free radical IC₅₀=6.23mg/m L,DPPH free radical IC₅₀=1.24mg/m L,ABTS free radical IC₅₀<0.5mg/m L,superoxide anion free radical IC₅₀=8mg/m L(2)Yeast active peptide has higher reducing power and better metal ion chelating ability.(3)Antioxidant peptides can effectively improve the survival rate of S.cerevisiae treated with 1?mol/L H₂O₂.(4)Antioxidant peptides can significantly reduce the intracellular lipid peroxide content of Saccharomyces cerevisiae damaged by H₂O₂oxidation,and can regulate the expression level of intracellular antioxidant enzymes in Saccharomyces cerevisiae.Yeast antioxidant peptide is a natural antioxidant with high antioxidant activity.3.Yeast active peptide interferes with the toxic effect of DON on pig intestinal epithelial cells through Keap1-Nrf2 antioxidant signaling pathway:Western blot blot hybridization technique was used to detect the antioxidant activity of yeast active peptide on Keap1-Nrf2 antioxidant pathway in DON cell injury model Protein expression level.The results show that:(1)Yeast active peptides can interfere with DON cytotoxicity to a certain extent and improve the survival rate of pig intestinal epithelial cells.(2)DON can cause a certain degree of oxidative damage to cells,and yeast active peptides can alleviate this oxidative damage and reduce the production of oxidized products.(3)The effect of DON can significantly reduce the content of glutathione and catalase in pig intestinal epithelial cells,and slightly increase the content of superoxide dismutase.The addition of yeast active peptides can significantly increase the expression of antioxidant enzymes in cells.(4)Yeast enzymatic hydrolysis active peptide can significantly upregulate nuclear factor-related factor 2 Nrf2,?phase detoxification enzyme heme oxygenase HO-1 and antioxidant quinone oxidoreductase NQO1 in pig intestinal epithelial cells damaged by DON oxidation Expression of related proteins in antioxidant signaling pathway.The above results indicate that a class of short peptides extracted from Brady yeast has good antioxidant activity,can scavenge most free radicals,and has good iron ion reducing ability and metal ion chelating ability,which is an excellent Natural antioxidants.Yeast active peptides have better growth promotion effect in organisms and can resist oxidative stress of Saccharomyces cerevisiae.In the DON-porcine intestinal epithelial cell model,the yeast active peptide can regulate the expression of antioxidant related proteins downstream of the Keap1-Nrf2 antioxidant signaling pathway by activating the body's antioxidant regulation mechanism,interfering with the DON-induced cellular oxidative stress response,and alleviating vomiting Toxic injury of pig intestinal epithelial cells.